α-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded fibril formation and toxicity.

α-Synuclein (α-syn) phosphorylation at serine 129 (pS129­α-syn) is substantially increased in Lewy body disease, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129­α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129­α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129­α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129­α-syn (WT­α-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129­α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129­α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129­α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129­α-syn as a measure of efficacy in clinical trials.

Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes.

Extracellular vesicles (EVs) have emerged as key players in cell-to-cell communication in both physiological and pathological processes in the Central Nervous System. Thus far, the intracellular pathways involved in uptake and trafficking of EVs within different cell types of the brain are poorly understood. In our study, the endocytic processes and subcellular sorting of EVs were investigated in primary glial cells, particularly linked with the EV-associated α-synuclein (α-syn) transmission. Mouse microglia and astrocytic primary cultures were incubated with DiI-stained mouse brain-derived EVs. The internalization and trafficking pathways were analyzed in cells treated with pharmacological reagents that block the major endocytic pathways. Brain-derived EVs were internalized by both glial cell types; however, uptake was more efficient in microglia than in astrocytes. Colocalization of EVs with early and late endocytic markers (Rab5, Lamp1) indicated that EVs are sorted to endo-lysosomes for subsequent processing. Blocking actin-dependent phagocytosis and/or macropinocytosis with Cytochalasin D or EIPA inhibited EV entry into glial cells, whereas treatment with inhibitors that strip cholesterol off the plasma membrane, induced uptake, however differentially altered endosomal sorting. EV-associated fibrillar α-Syn was efficiently internalized and detected in Rab5- and Lamp1-positive compartments within microglia. Our study strongly suggests that EVs enter glial cells through phagocytosis and/or macropinocytosis and are sorted to endo-lysosomes for subsequent processing. Further, brain-derived EVs serve as scavengers and mediate cell-to-glia transfer of pathological α-Syn which is also targeted to the endolysosomal pathway, suggesting a beneficial role in microglia-mediated clearance of toxic protein aggregates, present in numerous neurodegenerative diseases.

Screening for Novel Inhibitors of Amyloid Beta Aggregation and Toxicity as Potential Drugs for Alzheimer's Disease.

AD is the most common neurodegenerative disorder characterized by progressive memory impairment and cognitive deficits. The pathology of AD is still unclear; however, several studies have shown that the aggregation of the Aß peptide in the CNS is an exclusively pathological process involved in AD. Currently, there is no proven medication to cure or prevent the disease progression. Nevertheless, various therapeutic approaches for AD show only relief of symptoms and mostly work on cognitive recovery. However, one of the promising approaches for therapeutic intervention is to use inhibitors for blocking the Aß peptide aggregation process. Recently, herbal phenolic compounds have been shown to have a therapeutic property for treatment of AD due to their multifaceted action. In this study, we investigated the effectiveness of SA, Gn Rb1, and DMyr on inhibiting the aggregation and toxicity of Aß40 and Aß42 using different biochemical and cell-based assays. Our results showed that SA and DMyr inhibit Aß40 and Aß42 fibrillation, seeded aggregation, and toxicity. Gn Rb1 did not have any effect on the aggregation or toxicity induced by Aß40 and Aß42. Moreover, SA and DMyr were able to disaggregate the preformed fibrils. Overall, these compounds may be used alone or synergistically and could be considered as a lead for designing new compounds that could be used as effective treatment of AD and related disorders.

The promise of the TGF-ß superfamily as a therapeutic target for Parkinson's disease.

A large body of evidence underscore the regulatory role of TGF-ß superfamily in the central nervous system. Components of the TGF-ß superfamily modulate key events during embryonic brain development and adult brain tissue injury repair. With respect to Parkinson's disease (PD), TGF-ß signaling pathways are implicated in the differentiation, maintenance and synaptic function of the dopaminergic neurons, as well as in processes related to the activation state of astrocytes and microglia. In vitro and in vivo studies using toxin models, have interrogated on the dopaminotrophic and protective role of the TGF-ß superfamily members. The evolution of genetic and animal models of PD that more closely recapitulate the disease condition has made possible the dissection of intracellular pathways in response to TGF-ß treatment. Although the first clinical trials using GDNF did not meet their primary endpoints, substantial work has been carried out to reappraise the TGF-ß superfamily's clinical benefit.

A double-hit in vivo model of GBA viral microRNA-mediated downregulation and human alpha-synuclein overexpression demonstrates nigrostriatal degeneration.

Preclinical and clinical studies support a strong association between mutations in the GBA1 gene that encodes beta-glucocerebrosidase (GCase) (EC 3.2.1.45; glucosylceramidase beta) and Parkinson's disease (PD). Alpha-synuclein (AS), a key player in PD pathogenesis, and GBA1 mutations may independently and synergistically cause lysosomal dysfunction and thus, embody clinically well-validated targets of the neurodegenerative disease process in PD. However, in vivo models, recapitulating pathological features of PD that can be used to dissect the nature of the complex relationship between GCase and AS on the nigrostriatal axis, the region particularly vulnerable in PD, are direly needed. To address this, we implemented a bidirectional approach in mice to examine the effects of: 1) GCase overexpression (wild-type and mutant N370S GBA) on endogenous AS levels and 2) downregulation of endogenous GCase (Gba) combined with AS overexpression. Striatal delivery of viral-mediated GCase overexpression revealed minimal effects on cortical and nigrostriatal AS tissue levels and no significant effect on dopaminergic system integrity. On the other hand, microRNA (miR)-mediated Gba1 downregulation (miR Gba), combined with virus-mediated human AS overexpression (+AS), yields decreased GCase activity in the cortex, mimicking levels seen in GBA1 heterozygous carriers (30-40%), increased astrogliosis and microgliosis, decreased striatal dopamine levels (50% compared to controls) and loss of nigral dopaminergic neurons (~33%)- effects that were all reversible with miR rescue. Most importantly, the synergistic neurodegeneration of miR Gba + AS correlated with augmented AS accumulation and extracellular release in the striatum. Collectively, our results suggest that GCase downregulation alone is not sufficient to recapitulate key pathological features of PD in vivo, but its synergistic interplay with AS, via increased AS levels and extracellular release, drives nigrostriatal neurodegeneration. Furthermore, we report a novel double-hit GBA-AS model that can be used to identify putative mechanisms driving PD pathophysiology and can be subsequently used to test novel therapeutic approaches.

Structural and Biophysical Characterization of Stable Alpha-Synuclein Oligomers.

The aggregation of α-synuclein (α-syn) into neurotoxic oligomers and fibrils is an important pathogenic feature of synucleinopatheis, including Parkinson's disease (PD). A further characteristic of PD is the oxidative stress that results in the formation of aldehydes by lipid peroxidation. It has been reported that the brains of deceased patients with PD contain high levels of protein oligomers that are cross-linked to these aldehydes. Increasing evidence also suggests that prefibrillar oligomeric species are more toxic than the mature amyloid fibrils. However, due to the heterogenous and metastable nature, characterization of the α-syn oligomeric species has been challenging. Here, we generated and characterized distinct α-syn oligomers in vitro in the presence of DA and lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). HNE and ONE oligomer were stable towards the treatment with SDS, urea, and temperature. The secondary structure analysis revealed that only HNE and ONE oligomers contain ß-sheet content. In the seeding assay, both DA and ONE oligomers significantly accelerated the aggregation. Furthermore, all oligomeric preparations were found to seed the aggregation of α-syn monomers in vitro and found to be cytotoxic when added to SH-SY5Y cells. Finally, both HNE and ONE α-syn oligomers can be used as a calibrator in an α-syn oligomers-specific ELISA.

Changes in the cellular fatty acid profile drive the proteasomal degradation of α-synuclein and enhance neuronal survival.

Parkinson's disease is biochemically characterized by the deposition of aberrant aggregated α-synuclein in the affected neurons. The aggregation properties of α-synuclein greatly depend on its affinity to bind cellular membranes via a dynamic interaction with specific lipid moieties. In particular, α-synuclein can interact with arachidonic acid (AA), a polyunsaturated fatty acid, in a manner that promotes the formation of α-helix enriched assemblies. In a cellular context, AA is released from membrane phospholipids by phospholipase A2 (PLA2 ). To investigate the impact of PLA2 activity on α-synuclein aggregation, we have applied selective PLA2 inhibitors to a SH-SY5Y cellular model where the expression of human wild-type α-synuclein is correlated with a gradual accumulation of soluble oligomers and subsequent cell death. We have found that pharmacological and genetic inhibition of GIVA cPLA2 resulted in a dramatic decrease of intracellular oligomeric and monomeric α-synuclein significantly promoting cell survival. Our data suggest that alterations in the levels of free fatty acids, and especially AA and adrenic acid, promote the formation of α-synuclein conformers which are more susceptible to proteasomal degradation. This mechanism is active only in living cells and is generic since it does not depend on the absolute quantity of α-synuclein, the presence of disease-linked point mutations, the expression system or the type of cells. Our findings indicate that the α-synuclein-fatty acid interaction can be a critical determinant of the conformation and fate of α-synuclein in the cell interior and, as such, cPLA2 inhibitors could serve to alleviate the intracellular, potentially pathological, α-synuclein burden.

Quantitative determination of aloin, antioxidant activity, and toxicity of Aloe vera leaf gel products from Greece.

BACKGROUND: Aloe vera is a popular medicinal plant used widely by the cosmetic, pharmaceutical, and food industries. The A. vera leaf gel, which is used mostly for its positive effects on human health, contains over 75 different bioactive compounds, including aloin. Aloin is a toxic compound, and its content in A. vera leaf gel products depends on the different cultivation conditions and especially on leaf processing. RESULTS: In this study, A. vera leaf gel products, varied in terms of leaf processing, were analyzed using liquid chromatography for their aloin content, their antioxidant activity by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS·+ ) and the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH· ) antioxidant activity assays and their toxicity against Aliivibrio fisheri and SH-SY5Y cells. In the samples processed with industrial methods and in those filtered in the lab, the content of aloin was found below the limit (0.1 mg L-1 ) of the EU legislation however, the unprocessed and unfiltered samples were found to contain more than 10 mg L-1 . Antioxidant activity was estimated to vary from 1.64 to 9.21 µmol Trolox mL-1 for DPPH· and from 0.73 to 5.14 µmol Trolox mL-1 for ABTS·+ . Toxicity values on A. fisheri, expressed as the concentration at 50% loss of initial luminescence, ranged from 0.03 to 0.09 mg mL-1 . The cytotoxic study indicated that aloin A at low concentrations (1 and 10 µg mL-1 ) protects SH-SY5Y cells from toxicity induced by hydrogen peroxide. CONCLUSIONS: Consequently, the filtration process of A. vera leaf gels, either laboratory or industrial, resulted in aloin A content below the EU legislation detection limits. © 2020 Society of Chemical Industry.

Modulation of ß-glucocerebrosidase increases α-synuclein secretion and exosome release in mouse models of Parkinson's disease.

Glucocerebrosidase gene (GBA) mutations are the most common genetic contributor to Parkinson's disease (PD) and are associated with decreased glucocerebrosidase (GCase) enzymatic activity in PD. PD patients without GBA mutations also exhibit lower levels of GCase activity in the central nervous system suggesting a potential contribution of the enzyme activity in disease pathogenesis, possibly by alteration of lysosomal function. α-synuclein (ASYN), a protein with a central role in PD pathogenesis, has been shown to be secreted partly in association with exosomes. It is possible that a dysfunction of the endocytic pathway through GCase may result in altered exosome release of ASYN. The aim of this study was to examine whether manipulating GCase activity in vivo and in vitro could affect ASYN accumulation and secretion. GCase overexpression in vitro resulted in a significant decrease of exosome secretion. Chronic inhibition of GCase activity in vivo, by administration of the covalent inhibitor conduritol-B epoxide in A53T-synuclein alpha gene Tg mice significantly elevated intracellular oligomeric ASYN species. Importantly, GCase inhibition, induced a profound increase in the number of brain exosomes released, as well as exosome-associated ASYN oligomers. Finally, virus-mediated expression of mutant GBA in the mouse striatum increased ASYN secretion in the same region. Together, these results provide for the first time evidence that a decrease of GCase or overexpression of mutant GCase in a chronic in vivo setting can affect ASYN secretion. Such effects may mediate enhanced propagation of ASYN, driving pathology in GBA-associated PD.

Defective synaptic connectivity and axonal neuropathology in a human iPSC-based model of familial Parkinson's disease.

α-Synuclein (αSyn) is the major gene linked to sporadic Parkinson's disease (PD), whereas the G209A (p.A53T) αSyn mutation causes a familial form of PD characterized by early onset and a generally severe phenotype, including nonmotor manifestations. Here we generated de novo induced pluripotent stem cells (iPSCs) from patients harboring the p.A53T mutation and developed a robust model that captures PD pathogenic processes under basal conditions. iPSC-derived mutant neurons displayed novel disease-relevant phenotypes, including protein aggregation, compromised neuritic outgrowth, and contorted or fragmented axons with swollen varicosities containing αSyn and Tau. The identified neuropathological features closely resembled those in brains of p.A53T patients. Small molecules targeting αSyn reverted the degenerative phenotype under both basal and induced stress conditions, indicating a treatment strategy for PD and other synucleinopathies. Furthermore, mutant neurons showed disrupted synaptic connectivity and widespread transcriptional alterations in genes involved in synaptic signaling, a number of which have been previously linked to mental disorders, raising intriguing implications for potentially converging disease mechanisms.